EP2303312B1 - Vaccin antivariolique à adn et antigènes présents à l'intérieur capables de susciter une réponse immunitaire - Google Patents

Vaccin antivariolique à adn et antigènes présents à l'intérieur capables de susciter une réponse immunitaire Download PDF

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EP2303312B1
EP2303312B1 EP09820931.5A EP09820931A EP2303312B1 EP 2303312 B1 EP2303312 B1 EP 2303312B1 EP 09820931 A EP09820931 A EP 09820931A EP 2303312 B1 EP2303312 B1 EP 2303312B1
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antigen
dna
vacv
vaccine
seq
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EP2303312A4 (fr
EP2303312A2 (fr
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Ruxandra Draghia-Akli
Jon Prigge
David B. Weiner
Lauren A. Hirao
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University of Pennsylvania Penn
Inovio Pharmaceuticals Inc
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University of Pennsylvania Penn
Inovio Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • A61K39/285—Vaccinia virus or variola virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24151—Methods of production or purification of viral material

Definitions

  • the present invention relates to consensus smallpox antigens, nucleic acid constructs encoding such antigens, and vaccines made therefrom for generating an immune response against smallpox virus, and use of vaccines to protect mammals against smallpox virus.
  • the commercially approved smallpox vaccines available for limited use are the established Dryvax vaccine and the Acambis vaccine.
  • This Wyeth vaccine is a lyophilized preparation of live vaccinia virus (VACV) derived from calf lymph. Wyeth discontinued distribution of smallpox vaccine to civilians in 1983.
  • VACV live vaccinia virus
  • the Acambis vaccine is a live tissue culture adapted vaccine stock that still is associated with severe adverse effects in humans.
  • Cidofovir a licensed drug for the treatment of cytomegalovirus retinitis in AIDS patients has broad-spectrum activity against virtually all DNA viruses.
  • cidofovir demonstrated in vitro and in vivo activity in mice against a number of poxviruses including variola and monkeypox (MPXV).
  • MPXV variola and monkeypox
  • a single dose of cidofovir showed high efficacy in protecting mice from lethal respiratory infection with either vaccinia or cowpox, provided drug treatment is initiated within a few days after exposure.
  • Hooper et al., 2007, Vaccine, 25(10):1814-1823 discusses a smallpox DNA vaccine delivered by a skin electroporation device that protects mice against intranasal poxvirus challenge.
  • An aspect of the present invention comprises DNA vaccines that are capable of generating a protective immune response in mammals against a smallpox virus.
  • the DNA vaccine comprises a DNA plasmid comprising a nucleotide sequence that encodes VACV A27L antigen, a DNA plasmid comprising a nucleotide sequence that encodes VACV F9L antigen, a DNA plasmid comprising a nucleotide sequence that encodes VACV H3L antigen, a DNA plasmid comprising a nucleotide sequence that encodes VACV L1R antigen, a DNA plasmid comprising a nucleotide sequence that encodes VACV A33R antigen, a DNA plasmid comprising a nucleotide sequence that encodes VACV A56R antigen, a DNA plasmid comprising a nucleotide sequence that encodes VACV B5R antigen, and a DNA plasmid comprising a nucleotide sequence that encode
  • the DNA plasmid comprises a DNA plasmid encoding each of VACV MV antigens A27L, F9L, H3L and L1R; a DNA plasmid encoding each of VACV EV antigens A33R, A56R and B5R; and a DNA plasmid encoding A4L antigen.
  • Another aspect of the present invention relates to use of the vaccines in inducing a protective immune response in a mammal to smallpox virus, including a neutralizing antibody response.
  • the use can comprise injecting the vaccine into tissue of said mammal.
  • the methods further comprise the step of electroporating said tissue with an electroporating amount of electrical energy.
  • nucleic acid construct refers to the DNA or RNA molecules that comprise a nucleotide sequence that encodes protein.
  • the coding sequence, or “encoding nucleic acid sequence,” can include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered.
  • the term "expressible form” refers to nucleic acid constructs that contain the necessary regulatory elements operably linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.
  • constant current is used herein to define a current that is received or experienced by a tissue, or cells defining said tissue, over the duration of an electrical pulse delivered to same tissue.
  • the electrical pulse is delivered from the electroporation devices described herein and contemplated for use with the plasmids and vaccines described herein. This current remains at a constant amperage in said tissue over the life of an electrical pulse because the electroporation device provided herein has a feedback element, preferably having instantaneous feedback.
  • the feedback element can measure the resistance of the tissue (or cells) throughout the duration of the pulse and cause the electroporation device to alter its electrical energy output (e.g., increase voltage) so current in same tissue remains constant throughout the electrical pulse (on the order of microseconds), and from pulse to pulse.
  • the feedback element comprises a controller.
  • feedback or “current feedback” is used interchangeably and means the active response of the provided electroporation devices, which comprises measuring the current in tissue between electrodes and altering the energy output delivered by the EP device accordingly in order to maintain the current at a constant level.
  • This constant level is preset by a user prior to initiation of a pulse sequence or electrical treatment.
  • the feedback is accomplished by the electroporation component, e.g., controller, of the electroporation device, as the electrical circuit therein is able to continuously monitor the current in tissue between electrodes and compare that monitored current (or current within tissue) to a preset current and continuously make energy-output adjustments to maintain the monitored current at preset levels.
  • the feedback loop is instantaneous as it is an analog closed-loop feedback.
  • electro-kinetic enhancement refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and/or water to pass from one side of the cellular membrane to the other.
  • decentralized current is used herein to define the pattern of electrical currents delivered from the various needle electrode arrays of the electroporation devices described herein, wherein the patterns minimize, or preferably eliminate, the occurrence of electroporation related heat stress on any area of tissue being electroporated.
  • feedback mechanism refers to a process performed by either software or hardware (or firmware), which process receives and compares the impedance of the desired tissue (before, during, and/or after the delivery of pulse of energy) with a present value, preferably current, and adjusts the pulse of energy delivered to achieve the preset value.
  • impedance is used herein when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus enabling comparisons with the preset current.
  • the "feedback mechanism” is performed by an analog closed loop circuit.
  • adjuvant is used herein to mean any molecule added to the DNA vaccines described herein to enhance antigenicity of the VACV antigen encoded by the DNA plasmids and encoding nucleic acid sequences described hereinafter.
  • protective immune response is used herein to mean a combination of antibody response along with cellular immune response, and preferably neutralizing antibody response, which results from immunization with the DNA vaccines provided herein.
  • Consensus or “consensus sequences” or “consensus antigens”, used interchangeable to describe the preferred antigens of the present invention, refers to a synthetic sequence that is generated based on contemporary isolates of a particular virus.
  • the consensus may be genetically closer to current circulating viral strains than any given natural virus isolate.
  • global sequencing is generally conducted with viruses sampled during chronic infections instead of viruses sampled during acute infection, developing a consensus vaccine response on epitopes that for the most part have escaped may be a disadvantage. To minimize this disadvantage, one useful strategy for vaccine design would be to take early transmitter sequences into account.
  • the consensus becomes an effective approach to minimize the degree of sequence dissimilarity between a vaccine strain and contemporary circulating viruses is to create artificial sequences that are "central" to these viruses.
  • One design strategy is to use a consensus sequence derived from the most common amino acid in every position in an alignment. Such consensus can then elicit a broad immune response against various natural viral isolates and polymorphisms in combinations not found in any natural virus.
  • An aspect of the present invention comprises DNA vaccines that are capable of generating a protective immune response in mammals against a pox virus.
  • the pox virus is smallpox virus.
  • the DNA vaccine comprises a plasmid or plasmids capable of expressing a plurality of VACV MV antigens, a plasmid or plasmids capable of expressing a plurality of VACV EV antigens, and a plasmid capable of expressing A4L antigen.
  • Each of said antigens can be expressed by a single DNA plasmid (includes multiple encoding sequences) or by distinct DNA plasmids.
  • each distinct antigen will be expressed by a distinct DNA plasmid.
  • the VACV MV antigens comprise: A27L, F9L, H3L, or L1R, while the VACV EV antigens comprise: A33R, A56R, or B5R.
  • each of the DNA plasmids comprise consensus DNA sequences that encode said antigens.
  • the consensus DNA sequences that encode VACV MV antigens comprise: SEQ ID NO: 3 (A27L), SEQ ID NO: 11 (F9L), SEQ ID NO: 13 (H3L), or SEQ ID NO: 15 (L1R).
  • the consensus DNA sequences that encode VACV EV antigens comprise: SEQ ID NO: 5 (B5R), SEQ ID NO: 7 (A33R), or SEQ ID NO: 9 (A56R).
  • the consensus DNA sequences that encode A4L comprise: SEQ ID NO: 1.
  • the DNA plasmid capable of expressing a plurality of VACV MV antigens comprises encoding sequences that encode a protein having a sequence comprising: SEQ ID NO: 4 (A27L), SEQ ID NO: 12 (F9L), SEQ ID NO: 14 (H3L), or SEQ ID NO: 16 (L1R)
  • the DNA plasmid capable of expressing a plurality of VACV MV antigens comprises encoding sequences that encode a protein having a sequence comprising: SEQ ID NO: 6 (B5R), SEQ ID NO: 8 (A33R), or SEQ ID NO: 10 (A56R)
  • DNA plasmid capable of expressing A4L antigen comprises encoding sequences that encode a protein having sequence of SEQ ID NO: 2.
  • the DNA vaccine comprises a plurality of distinct DNA plasmids that comprise encoding DNA sequences: SEQ ID NO: 1 (A4L), SEQ ID NO: 3 (A27L), SEQ ID NO: 5 (B5R), SEQ ID NO: 7 (A33R), SEQ ID NO: 9 (A56R).SEQ ID NO: 11 (F9L), SEQ ID NO: 13 (H3L), and SEQ ID NO: 15 (L1R), respectively.
  • the DNA vaccine comprises a plurality of distinct DNA plasmids that comprise encoding DNA sequences that encode a protein having sequences: SEQ ID NO: 2 (A4L), SEQ ID NO: 4 (A27L), SEQ ID NO: 6 (B5R), SEQ ID NO: 8 (A33R), SEQ ID NO: 10 (A56R).SEQ ID NO: 12 (F9L), SEQ ID NO: 14 (H3L), and SEQ ID NO: 16 (L1R), respectively.
  • the consensus encoding sequences are human codon-optimized.
  • the DNA vaccine comprises DNA plasmids pGX4001, pGX4002, pGX4003, pGX4004, pGX4005, pGX4006, pGX4007, or pGX4008, or a combination thereof.
  • the vaccines may be for injection into tissue of a mammal.
  • the pox virus is smallpox virus.
  • the injection is intradermal or intramuscular injection.
  • the use can further comprise the step of electroporating said tissue with an electroporating amount of electrical energy.
  • the electroporating step comprises delivering a constant current to said tissue. More preferably, the electroporating step comprises delivering 0.2 A of current.
  • the use comprises repeating said injecting step.
  • the delivering step comprises delivering eight distinct DNA plasmids.
  • the DNA vaccine described herein is formulated using DNA plasmid formulations that have a high DNA concentration.
  • the high DNA concentration can be a concentration of 5 mg/mL or more, 6 mg/mL or more, 7 mg/mL or more, 8 mg/mL or more, 9 mg/mL or more, 10 mg/mL or more, 11 mg/mL or more, 12 mg/mL or more, 13 mg/mL or more, 14 mg/mL or more, 15 mg/mL or more.
  • the plasmid DNA may be in a concentration of 5-15 mg/mL, 5-14 mg/mL, 5-13 mg/mL, 5-12 mg/mL, 5-11 mg/mL, 5-10 mg/mL, 5-9 mg/mL, 5-8 mg/mL, a concentration of 6-15 mg/mL, 6-14 mg/mL, 6-13 mg/mL, 6-12 mg/mL, 6-11 mg/mL, 6-10 mg/mL, 6-9 mg/mL, 6-8 mg/mL, a concentration of 7-15 mg/mL, 7-14 mg/mL, 7-13 mg/mL, 7-12 mg/mL, 7-11 mg/mL, 7-10 mg/mL, 7-9 mg/mL, 8-15 mg/mL, 8-14 mg/mL, 8-13 mg/mL, 8-12 mg/mL, 8-11 mg/mL, 8-10 mg/mL, 9-15 mg/mL, 9-14 mg/mL, 9-13 mg/mL, 9-12 mg/mL, 9-14
  • each distinct DNA plasmid is present at a high dose, which is a dose: greater than 50 ⁇ g, greater than 60 ⁇ g, greater than 70 ⁇ g, greater than 80 ⁇ g, greater than 90 ⁇ g, greater than 100 ⁇ g, greater than 110 ⁇ g, greater than 120 ⁇ g, greater than 130 ⁇ g, greater than 140 ⁇ g, greater than 150 ⁇ g, greater than 160 ⁇ g, greater than 170 ⁇ g, greater than 180 ⁇ g, greater than 190 ⁇ g, greater than 200 ⁇ g, greater than 210 ⁇ g, greater than 220 ⁇ g, greater than 230 ⁇ g, greater than 240 ⁇ g, or greater than 250 ⁇ g.
  • the high dose is greater than 120 ⁇ g, and more preferably 125 ⁇ g.
  • the DNA vaccines can further include an adjuvant.
  • the adjuvant is selected from the group consisting of: alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNF ⁇ , TNF ⁇ , GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80,CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE.
  • genes which may be useful adjuvants include those encoding: MCP-1, MIP-1 ⁇ , MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p
  • the adjuvant is selected from IL-8, IL-12, IL-15, IL-18, IL-28, MCP-1, MIP-1 ⁇ , MIP-1p, RANTES, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, CTACK, TECK, or MEC, or a combination thereof; and more preferably, the adjuvant is IL-12, IL-15, IL-28, or RANTES.
  • Poxviruses are a large complex viruses from the family Poxviridae and include VACV and variola virus (smallpox). Four genera of poxviruses are known to infect humans, and include orthopox, parapox, yatapox, molluscipox.
  • Orthopox variola virus, vaccinia virus, cowpox virus, monkeypox virus, smallpox (eradicated); Parapox: orf virus, pseudocowpox, bovine papular stomatitis virus; Yatapox: tanapox virus, yaba monkey tumor virus; Molluscipox: molluscum contagiosum virus (MCV).
  • poxviruses include Orthopoxvirus, such as camelpox virus, cowpox virus, ectromelia virus, monkeypox virus, raccoon poxvirus, skunk poxvirus, Tatera poxvirus, Uasin Gishu virus, variola virus, Volepox virus, Parapoxvirus such as Ausdyk virus, Bovin papular stomatitis virus, orf virus, pseudocowpox virus, red deer poxvirus, seal parapoxvirus, Capripoxvirus such as sheep-pox virus, goatpox Virus lumpyskin disease virus, Suipoxvirus such as swinepox virus, Leporipoxvirus such as myxoma virus fibroma virus, hare fibroma virus, squirrel fibroma virus, western squirrel fibroma, Avipoxvirus of many species, Yatapoxvirus such as Tantpox virus, Yabapoxvirus, Molluscip
  • Routes of administration include, but are not limited to, intramuscular, intranasally, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterially, intraoccularly and oral as well as topically, transdermally, by inhalation or suppository or to mucosal tissue such as by lavage to vaginal, rectal, urethral, buccal and sublingual tissue.
  • Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection.
  • Genetic constructs may be administered by means including, but not limited to, traditional syringes, needleless injection devices, "microprojectile bombardment gone guns", or other physical methods such as electroporation ("EP”), "hydrodynamic method", or ultrasound.
  • electroporation devices and electroporation methods preferred for facilitating delivery of the DNA vaccines of the present invention include those described in U.S. Patent No. 7,245,963 by Draghia-Akli, et al. , U.S. Patent Pub. 2005/0052630 submitted by Smith, et al. Also preferred, are electroporation devices and electroporation methods for facilitating delivery of the DNA vaccines provided in co-pending and co-owned U.S. Patent Application, Serial No. 11/874072, filed October 17, 2007 , which claims the benefit under 35 USC 119(e) to U.S. Provisional Applications Ser. Nos. 60/852,149, filed October 17, 2006 , and 60/978,982, filed October 10, 2007 .
  • electroporation devices can be configured to deliver to a desired tissue of a mammal a pulse of energy producing a constant current similar to a preset current input by a user.
  • the electroporation device comprises an electroporation component and an electrode assembly or handle assembly.
  • the electroporation component can include and incorporate one or more of the various elements of the electroporation devices, including: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch.
  • the electroporation component can function as one element of the electroporation devices, and the other elements are separate elements (or components) in communication with the electroporation component. In some embodiments, the electroporation component can function as more than one element of the electroporation devices, which can be in communication with still other elements of the electroporation devices separate from the electroporation component.
  • the present invention is not limited by the elements of the electroporation devices existing as parts of one electromechanical or mechanical device, as the elements can function as one device or as separate elements in communication with one another.
  • the electroporation component is capable of delivering the pulse of energy that produces the constant current in the desired tissue, and includes a feedback mechanism.
  • the electrode assembly includes an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives the pulse of energy from the electroporation component and delivers same to the desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during delivery of the pulse of energy and measures impedance in the desired tissue and communicates the impedance to the electroporation component.
  • the feedback mechanism can receive the measured impedance and can adjust the pulse of energy delivered by the electroporation component to maintain the constant current.
  • the plurality of electrodes can deliver the pulse of energy in a decentralized pattern. In some embodiments, the plurality of electrodes can deliver the pulse of energy in the decentralized pattern through the control of the electrodes under a programmed sequence, and the programmed sequence is input by a user to the electroporation component. In some embodiments, the programmed sequence comprises a plurality of pulses delivered in sequence, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes with one neutral electrode that measures impedance, and wherein a subsequent pulse of the plurality of pulses is delivered by a different one of at least two active electrodes with one neutral electrode that measures impedance.
  • the feedback mechanism is performed by either hardware or software.
  • the feedback mechanism is performed by an analog closed-loop circuit.
  • this feedback occurs every 50 ⁇ s, 20 ⁇ s, 10 ⁇ s or 1 ⁇ s, but is preferably a real-time feedback or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time).
  • the neutral electrode measures the impedance in the desired tissue and communicates the impedance to the feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the pulse of energy to maintain the constant current at a value similar to the preset current.
  • the feedback mechanism maintains the constant current continuously and instantaneously during the delivery of the pulse of energy.
  • a pharmaceutically acceptable excipient can include such functional molecules as vehicles, adjuvants, carriers or diluents, which are known and readily available to the public.
  • the pharmaceutically acceptable excipient is an adjuvant or transfection facilitating agent.
  • the nucleic acid molecule, or DNA plasmid is delivered to the cells in conjunction with administration of a polynucleotide function enhancer or a genetic vaccine facilitator agent (or transfection facilitating agent).
  • Polynucleotide function enhancers are described in U.S. Serial Number 5,593,972 , 5,962,428 and International Application Serial Number PCT/US94/00899 filed January 26, 1994 .
  • Genetic vaccine facilitator agents are described in US. Serial Number 021,579 filed April 1, 1994 .
  • the transfection facilitating agent can be administered in conjunction with nucleic acid molecules as a mixture with the nucleic acid molecule or administered separately simultaneously, before or after administration of nucleic acid molecules.
  • transfection facilitating agents includes surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct.
  • ISCOMS immune-stimulating complexes
  • LPS analog including monophosphoryl lipid A
  • muramyl peptides muramyl peptides
  • quinone analogs and vesicles such as squalene and squalene
  • hyaluronic acid may also be used administered in conjunction with the genetic construct.
  • the DNA plasmid vaccines may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example WO9324640 ), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.
  • the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid.
  • the DNA plasmids are delivered with an adjuvant that are genes for proteins which further enhance the immune response against such target proteins.
  • genes are those which encode other cytokines and lymphokines such as alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNF ⁇ , TNF ⁇ , GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80,CD86 and IL-15 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE.
  • cytokines and lymphokines such as alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNF ⁇ , TNF ⁇ , GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12,
  • genes which may be useful include those encoding: MCP-1, MIP-1 ⁇ , MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38,
  • the DNA plasmid vaccines according to the present invention comprise DNA quantities of from about 1 microgram to about 10 milligrams, about 10 microgram to about 10 milligrams, about 100 microgram to about 10 milligrams, about 200 microgram to about 10 milligrams, about 300 microgram to about 10 milligrams, about 400 microgram to about 10 milligrams, about 500 microgram to about 10 milligrams, about microgram to about 1 milligrams, about 10 microgram to about 1 milligrams, about 100 microgram to about 1 milligrams, about 200 microgram to about 1 milligrams, about 300 microgram to about 1 milligrams, about 400 microgram to about 1 milligrams, about 500 microgram to about 1 milligrams, about 100 microgram to about 1 milligrams, about 200 microgram to about 1 milligrams, about 300 microgram to about I milligrams, about 400 microgram to about 1 milligrams, or about 500 microgram to about 1 milligrams.
  • the quantity of DNA present comprise
  • DNA plasmid vaccines according to the present invention are formulated according to the mode of administration to be used. In cases where DNA plasmid vaccines are injectable compositions, they are sterile, and/or pyrogen free and/or particulate free.
  • An isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. In some embodiments, a stabilizing agent that allows the formulation to be stable at room or ambient temperature for extended periods of time, such as LGS or other polycations or polyanions is added to the formulation.
  • the use of vaccines of the invention to elicit an immune response in mammals against a consensus smallpox antigen include inducing mucosal immune responses.
  • This can be achieved using one or more of CTACK protein, TECK protein, MEC protein and functional fragments thereof or expressible coding sequences thereof in combination with an DNA plasmid including a consensus smallpox antigen, described above.
  • the one or more of CTACK protein, TECK protein, MEC protein and functional fragments thereof may be for administration prior to, simultaneously with or after administration of the DNA plasmid smallpox vaccines provided herein.
  • an isolated nucleic acid molecule that encodes one or more proteins of selected from the group consisting of: CTACK, TECK, MEC and functional fragments thereof is for administration to the mammal.
  • the DNA formulations for use with a muscle or skin EP device described herein have high DNA concentrations, preferably concentrations that include microgram to tens of milligram quantities, and preferably milligram quantities, of DNA in small volumes that are optimal for delivery to the skin, preferably small injection volume, preferably 25-200 microliters ( ⁇ L).
  • the DNA formulations have high DNA concentrations, such as 1 mg/mL or greater (mg DNA/volume of formulation). More preferably, the DNA formulation has a DNA concentration that provides for gram quantities of DNA in 200 ⁇ L of formula, and more preferably gram quantities of DNA in 100 ⁇ L of formula.
  • the DNA plasmids for use with the EP devices of the present invention can be formulated or manufactured using a combination of known devices and techniques, but preferably they are manufactured using an optimized plasmid manufacturing technique that is described in a commonly owned, co-pending U.S. provisional application U.S. Serial No. 60/939,792 , which was filed on May 23, 2007.
  • the DNA plasmids used in these studies can be formulated at concentrations greater than or equal to 10 mg/mL.
  • the manufacturing techniques also include or incorporate various devices and protocols that are commonly known to those of ordinary skill in the art, in addition to those described in U.S. Serial No. 60/939792 , including those described in a commonly owned patent, US Patent No.
  • VACV genes A4L, A27L, A33R, A56R, B5R, F9L, H3L, and L1R (from the Western Reserve Strain), were chemically synthesized from synthetic oligonucleotides, human codon -optimized, and modified to contain a Kozak consensus sequence and IgE leader sequence at the 5'end and HA epitope tag at the 3'end of the DNA sequence.
  • modified gene cassettes were cloned using conventional cloning methods into the eukaryotic expression plasmid, pVAX1 (Invitrogen, Carlsbad, CA) by GENEART (Burlingame, CA).
  • each gene is regulated by the CMV promoter.
  • the synthetic gene cassettes of A4L and B5R were cloned into the HindIII and XhoI sites to make the expression plasmids pGX4001 and pGX4003, respectively.
  • pGX4007 and pGX4008 were prepared by cloning the synthetic gene cassettes of H3L and L1R into the HindIII and BamHI restriction sites.
  • the remaining expression plasmids, pGX4002 and pGX4006 were made by cloning the synthetic gene cassettes of A27L and F9L into the KpnI/XhoI and EcoRI/XbaI restriction sites, respectively. After cloning, all antigens were confirmed by sequencing.
  • Plasmids were manufactured to high concentrations and purified using the manufacturing procedure described by Hebel et al. in US7,238,522 with modifications. This method yields endotoxin-free plasmid formulations ( ⁇ 10 EU/mg) at very high plasmid concentrations, adapted for biopharmaceutical delivery of vaccines. All plasmid preparations were formulated and prepared with 1% weight/weight with high-performance liquid chromatography (HPLC) purified low molecular weight poly-L-glutamate (LGS, average MW 10,900) in sterile water.
  • HPLC high-performance liquid chromatography
  • All plasmids (pGX4001 to pGX4008) were combined to make a single vaccine preparation consisting of 125 ⁇ g of each plasmid in a total volume of 0.1 mL for the ID or 0.5 mL for the IM administration.
  • PBMC Plasma samples were bled every two weeks during the vaccination schedule and every three post-challenge. Animals were anesthetized intramuscularly with ketamine HCL (10 to 30 mg/kg). Blood was collected in EDTA tubes. PBMC's were isolated from whole blood by standard Ficoll-Hypaque density gradient centrifugation, resuspended in complete culture medium (RPMI 1640 with 2mM L-glutamine supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin, and 55 ⁇ M ⁇ -mercaptoethanol).
  • RPMI 1640 with 2mM L-glutamine supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin, and 55 ⁇ M ⁇ -mercaptoethanol.
  • Antigen-specific ELISA To determine the IgG antibody responses, ELISAs were performed by coating a MaxiSorp Immuno 96 well plate (Nunc, Rochester, NY) with 50 ng of purified antigen (A4L, A27L, A33R, A56R, B5R, F9L, H3L, or L1R) diluted in PBS. After overnight incubation at 4°C, plates were washed with PBS supplemented with 0.05% Tween 20 (PBS-T) and then blocked for 1 h at RT with PBS supplemented with 3% BSA.
  • purified antigen A4L, A27L, A33R, A56R, B5R, F9L, H3L, or L1R
  • Serum collected from individual Cynomolgus macaques were diluted in PBS supplemented with 0.5% BSA; 0.05% Tween 20, and incubated overnight at 4°C with 50 ⁇ l of the diluted serum.
  • the wells were washed with PBS-T and then incubated with the secondary antibody, goat anti-rabbit IgG-HRP conjugated (Sigma-Aldrich, St. Louis, MO) and diluted 1 in 10,000 in PBS supplemented with 0.5% BSA; 0.05% Tween 20 (100 ⁇ L/well). The wells were incubated for 1 h at room temperature and washed. TMB substrate and stop solution was added to each well according to manufacturer's recommendations (KPL, Gaithersburg, MD).
  • Absorbance was measured at 450 nm using the Lumistar Galaxy plate reader (BMG Labtech). Endpoint titers are expressed as the reciprocal of the highest serum dilution yielding a positive reactivity greater than two-fold above negative control serum.
  • VACV ELISA Microtiter plates were coated with paraformaldehyde-fixed sucrose-gradient purified VACV WR strain (Advanced Biotechnologies, Inc.) at a concentration of 0.6 ⁇ g/ml and incubated overnight at 4°C. Plates were blocked for 2 h at 37°C using PBS-T supplemented with 5% non-fat dry milk (PBS-TM). Wells were washed 8 times with PBS-T and incubated with serial dilutions of monkey serum for 1 h at 37°C. After washing, the wells were incubated with secondary antibody, with goat anti-monkey IgG conjugated to horseradish peroxidase (KPL), and ABTS substrate (Sigma-Aldrich). The reaction was stopped with the addition of 100 ⁇ L of 10% SDS and read at 405 nm using a Molecular Devices SpectraMax Plus 384.
  • Endpoint titers are expressed as the reciprocal of the highest serum dilution yielding a positive reactivity ⁇ mean O.D. plus 3 S.D. of the negative control serum.
  • the peptides used for this study were derived from the coding region of A4L, A27L, A33R, A56R, B5R, F9L, H3L, and L1R of WR strain of VACV.
  • Total antigen peptide libraries were synthesized for A4L, A27L, A33R, A56R, F9L, H3L, and L1R. All peptides were 15-mers overlapping by either 9 amino acids (in the case of A27L), 11 amino acids (in the case of A4L, A33R, A56R, F9L, H3L, and L1R), or 6 amino acids (in the case of B5R).
  • the A27L library was prepared by Invitrogen. All other libraries were prepared by GenScript Corporation (Piscataway, NJ). Libraries were prepared as the corresponding peptide pool at a concentration of 10 mg/mL in DMSO.
  • IFN- ⁇ ELISPOT assay The nonhuman primate ELISpot assays were performed (See Boyer,J.D. et al.. J. Med Primatol. 34, 262-270 (2005 ). Antigen-specific responses were determined by subtracting the number of spots in the negative control wells from the wells containing peptides. Results are shown as the mean value (spots/million splenocytes) obtained for triplicate wells.
  • Carboxyfluorescein succinimidyl ester (CFSE) conjugation and flow cytometry analysis of PBMCs Cells were pelleted and resuspended in 1 ml carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (Molecular Probes, Eugene, OR) in PBS (1:2000 dilution). Cells were incubated at 37°C for 10 min. Cells were washed with complete media and resuspended to a concentration of 1x10 6 cells/100 ⁇ l and plated in 96 well round bottom plates with 100 ⁇ l of total peptide pools. Five ⁇ g/ml Concavalin A (positive) and complete media (negative) were used as controls.
  • CFDA-SE carboxyfluorescein diacetate succinimidyl ester
  • Flow cytometry data was analyzed using FlowJo software (Tree Star, Ashland, OR), gating on CD3 + lymphocytes. Thirty to fifty thousand CD3 + lymphocytes were collected per sample. Data is shown after media subtraction. Proliferative responses to A4 were not assessed due to high background proliferation in pre-immune samples.
  • Antibody Reagents Directly conjugated antibodies were obtained from the following: BD Biosciences (San Jose, CA): IL-2 (PE), CD3 (APC Cy7), CD8 (APC), IFN- ⁇ (Alexa Fluor 700), and TNF- ⁇ (PE Cy7), CD95 (PE Cy5) and CD4 (PerCP Cy5.5).
  • CD28 (ECD) was obtained from Beckman Coulter.
  • PBMCs were resuspended to 1 x 10 6 cells/100 ⁇ l in complete RPMI and plated in 96 well plates with A27L and B5R stimulating peptides 100 ⁇ l of 1:200 dilutions.
  • An unstimulated and positive control Staphylococcus enterotoxin B, 1 ⁇ g/ml; Sigma-Aldrich
  • Staphylococcus enterotoxin B 1 ⁇ g/ml
  • Sigma-Aldrich was included in each assay.
  • Cells were incubated for 5 hours at 37°C. Following incubation, the cells were washed (PBS) and stained with surface antibodies. The cells were washed and fixed using the Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA) according to instructions. Following fixation, the cells were washed twice in the perm buffer and stained with antibodies against intracellular markers. Following staining, the cells were washed, fixed (PBS containing 1% paraform
  • Flow cytometry Cells were analyzed on a modified LSR II flow cytometer (BD Immunocytometry Systems, San Jose, CA). Fifty thousand CD3 + events were collected per sample. Data analysis was performed using FlowJo version 8.6.3 (TreeStar, San Carlos, CA). Initial gating used a forward scatter area (FSC-A) versus height (FSC-H) plot to remove doublets. The events were subjected to a lymphocyte gate by a FSC-A versus SSC plot. Live T cells were identified by a live/dead versus CD3 + plot. Following this, events are sequentially gated on CD8 + and CD4- events versus IFN- ⁇ to account for down-regulation.
  • FSC-A forward scatter area
  • FSC-H height
  • the Zaire strain, V79-I-005 monkeypox virus Master Seed NR-523
  • monkeypox virus was obtained from the National Institutes of Health Biodefense and Emerging Infections Research Resources Repository. This Zaire strain was originally obtained from a fatally infected human from Zaire in 1979 (isolated by World Health Organization Collaborating Center for Smallpox and Other Poxvirus Infections at the US Center for Disease Control and Prevention).
  • the inoculum was prepared by passaging the virus in chicken embryo fibroblasts, and purified by sedimentation through a standard sucrose gradient. It was propagated in chicken embryo fibroblasts and purified.
  • Monkeypox virus challenge Four weeks after the last vaccination (day 91), Cynomolgus macaques were anesthetized as described herein and intravenously infused with 2 x 10 7 PFU of monkeypox virus NR-523 into the saphenous vein using a 23 gauge butterfly. In order to confirm the actual delivered dose, the challenge inoculum was back-tittered on Vero E6 cells using standard plaque assay technique.
  • Real-time PCR to detect monkeypox virus genomes.
  • DNA was extracted from frozen blood samples using QIAamp DNA mini kit (Qiagen).
  • the real-time PCR was prepared according to manufacturer's instructions for Quantitative Pan-orthopox HA PCR assay (Applied Biosystems) and consisted of the following primers for amplification hemagglutinin gene: OPHA F89: 5'-ATGTACTATCTCAACGTAGTAG-3' (SEQ ID NO.: 17) and OPHA R219: 5'-CTGCAGAACATAAAACTATTAATATG-3' (SEQ ID NO.:18).
  • the TaqMan probe (OPHA P143S-MGB: 6FAM AGTGCTTGGTATAAGGAG MGBNFQ (SEQ ID NO.:19 and SEQ ID .: 20)) was FAM-labeled at the 5'-end and contained a nonfluorescent quencher. Viral genome copies were performed with a LightCycler 1.5 (Roche).
  • VACV-neutralizing antibody Serum from monkeys were collected throughout the duration of the study, heat-inactivated (56°C for 30 min), and evaluated for the presence of VACV-neutralizing antibodies using classical plaque reduction neutralization tests. Each assay included as a positive control FDA standard reference Vaccinia Ig (Cangene) and validated at the Center for Biologies Evaluation and Research at the FDA. Negative controls included serum from unvaccinated macaques. One to four serial dilutions of the serum were prepared in complete culture medium and added to 24 well plates of Vero E6 cells (100% confluent) in triplicate. 4.5 x 10 5 PFU of Zaire 79 strain was added to each well of a 24-well plate.
  • a 500 ⁇ L semisolid overlay of pre-warmed methylcellulose (comprised of equal volume of 4% MEM (supplemented 4% FBS, 4 mM L-glutamine and 1% methylcellulose) was added to each well. Plates were incubated at 37°C; 5% CO2 for 72 h. Cell monolayers were stained with 250 ⁇ L of 0.1% crystal violet staining solution (prepared in 20% methanol).
  • Plaques were counted and percent neutralization was calculated relative to the number of plaques in the absence of antibody. Titers represent the reciprocal of the highest dilution resulting in 50% reduction in the number of plaques.
  • CBC Complete blood count analysis
  • Hematology parameters included: hematocrit, hemoglobin, total leukocyte count, and differential leukocyte counts (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelet count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular and hemoglobin concentration.
  • Each gene was synthetically-constructed and prepared by GeneArt Inc. (Toronto, ON) from oligonucleotides.
  • the oligonucleotides were codon-optimized from the Vaccinia Virus Western Reserve (WR) strain and cloned into pVAX1 (Invitrogen, Carlsbad, CA) using standard cloning methods.
  • the DNA vaccine plasmids, pGX4001 and pGX4003, encoding the optimized genes for A4L and B5R, respectively, were prepared by cloning the synthetically-constructed fragment into the Hind III and Xho I restriction sites.
  • pGX4004 (encoding A33R) and pGX4005 (encoding A56R) were prepared by cloning the DNA fragment into the Hind III and Xba I restriction sites.
  • pGX4007 and pGX4008 were prepared by cloning the DNA fragment encoding H3L and L1R, respectively, into the Hind III and Bam HI restriction sites.
  • F9L pGX4006
  • A27L pGX4002
  • a Kozak consensus sequence and an IgE leader sequence was added to the 5'-end of each gene.
  • a HA-epitope tag was added to the 3'-end of the gene.
  • plasmids were produced using partially the manufacturing procedure described by Hebel et al. in US7,238,522 with modifications, which yielded plasmid formulations at very high plasmid concentrations, adapted for biopharmaceutical delivery of vaccines (for example purposes see Figure 1 ).
  • a smallpox multivalent DNA vaccine composed of the VACV antigens A4L, A27, A33R, A56R, B5R, F9L, H3L, and L1R was produced.
  • RNA, protein and endotoxin demonstrated high purity with undetectable RNA, protein and endotoxin, average concentration of 10.7 ⁇ 0.7 mg/mL and supercoiled percentage of 94.5 ⁇ 1.1% after storage at -80°C for over 1 year (data not shown).
  • Each antigen in the vaccine preparation elicited both a robust antibody and cellular immune response in mice or rabbits (data not shown).
  • IMV F9L 23.8 Membrane glycoprotein structurally related to L1R and involved in cell fusion/entry IMV H3L 37.5 C-terminal transmembrane protein, morphogenesis, neutralizing antibodies.
  • EEV A56R 69-85 Encodes the Hemagglutinin gene and involved in cell fusion EEV B5R 35.1 Type I membrane, viral egress, target of neutralizing antibodies.
  • endotoxin-free plasmid preparations was diluted in sterile water and formulated at 1% weight/weight with high-performance liquid chromatography (HPLC) purified low molecular weight poly-L-glutamate (LGS, average MW 10,900), as previously described in Draghia-Akli R, Khan AS, Pope MA, Brown PA.
  • HPLC high-performance liquid chromatography
  • plasmid-based therapies For these plasmid-based therapies to be effectively transferred to humans, it is preferred to have larger quantities of plasmid in a small formulation volume (volumes that are similar to that of classic vaccines). Furthermore, the transgene product should be secreted efficiently from the target organ, and be detectable and active.
  • a constant current electroporation device for ID application (CELLECTRA®, VGX Pharmaceuticals, Inc., Blue Bell, PA) was developed with micro-electrodes (mEP) mounted on a sterilizable, disposable plastic array, which is the only component that actually touches the skin of the patient (to prevent cross-contamination).
  • the concentrated, high purity vaccine formulation in a small volume (volumes similar to that of classic vaccines, i.e., between 50 and 300 ⁇ L, and more preferably between 50-100 ⁇ L or 100-200 ⁇ L) is delivered to the selected area, the target area, and then the target area is surrounded by the micro-array.
  • the micro-electrodes are inserted into the skin.
  • the plastic array creates a uniform pressure around the micro-electrodes inserted into the skin, which helps to generate a uniform electric field during the EP process in the target area.
  • the plasmids were administered via a single intramuscular (IM) injection of 800 ⁇ g (100 ⁇ g each antigen and/or empty vector up to 800 ⁇ g per injection) in the following combinations: Group 1 rabbits were immunized with a combination of eight plasmids expressing varying smallpox antigens (A13L, A14L, A27L, A33R, B5R, D8L, H3L, L1R); group 2 rabbits were immunized with a combination of four different antigens (A27L, B5R, D8L, L1R); group 3 received an immunization with an individual plasmid expressing a single antigen (B5R).
  • IM intramuscular
  • plasmids were administered into semimembranosus muscle followed by electroporation using the CELLECTRA ® device (VGX Pharmaceuticals, Inc., Blue Bell, PA), at 0.6 Amps, 3 pulses, 52 ms/pulse, 1 sec between pulses. Sera were collected from rabbits at different time points and used to determine antibody responses by protein ELISA.
  • Antibody responses were improved by electroporation in both immunized groups irrespective of the number of antigens delivered.
  • Humoral responses to various antigens were also measured by ELISpot: B5R ( Figure 11 ), H3L ( Figure 12 ), A27L ( Figure 13 ), L1R ( Figure 14 ).
  • Sera from groups 1 and 2 demonstrated significant response to A27L and L1R (*p ⁇ 0.05 compared to Group 3), and the immune response to a single vaccine was not affected when these antigens were administered in groups of either 4 or 8 antigens.
  • ELISpot results 2 weeks after the first immunization showed that the group that received the IM injection and electroporation had, on average, 2.5x as many interferon-gamma (IFN ⁇ ) producing cells than the groups receiving the IM injection alone.
  • IFN ⁇ interferon-gamma
  • a combination vaccine consisting of several different Vaccinia Virus antigens was evaluated to determine the efficacy of the combination vaccine as measured by the level of antibody responses generated in the animals.
  • the DNA delivery method comparing the delivery either intradermally (ID) or intramuscularly (IM), was evaluated for efficacy.
  • the DNA was delivered using the CELLECTRA ® constant current device.
  • DNA vaccines which included plasmids encoding various vaccinia virus antigens, were administered (Day 0, 21, and 35) either intradermally (ID) (100 ⁇ l) or intramuscularly (IM) (500 ⁇ l or 1000 ⁇ l), and each vaccine consisted of a total of 1 mg of total plasmid per rabbit (250 ⁇ g per antigen).
  • the plasmid combination utilized in the vaccine formulations consisted of: influenza H5 hemagglutinin-expressing plasmid (H5HA) (used as positive control for the assay), and a combination of three vaccinia virus antigens (A4L, A27L, and B5R; see Example 1, above).
  • H5HA influenza H5 hemagglutinin-expressing plasmid
  • A4L, A27L, and B5R see Example 1, above.
  • Each DNA vaccine formulation was prepared in 1% LGS.
  • All DNA vaccines were administered into semimembranosus muscle followed by electroporation using the CELLECTRA ® constant current device using a variety of electroporation conditions as shown in Figure 18 .
  • Groups A to D were subjected to an ID injection of 100 ⁇ l and electroporated at 0.2 Amps and either received 2 pulses (Group A), 3 pulses (Group B), 4 pulses (Group C), 6 pulses (Group D).
  • Group E received an ID injection, but did not receive an electroporation.
  • Group F, G and 1 were intramuscularly (IM) administered with 500 ⁇ l formulation of the DNA vaccine and were electroporated at 0.5 Amps and received 3 pulses, with each consisting of either an 80, 4, 4, and 10-15 sec lag period, respectively.
  • IM intramuscularly
  • Group J received no electroporation (IM, 500 ⁇ l).
  • Group H received 1000 ⁇ l of an intramuscular injection and electroporation conditions consisting of 0.5 Amps, 4 sec lag, and 3 pulses.
  • the CELLECTRA ® constant current device was programmed to deliver 52 ms/pulse and 1 sec between pulses. Sera was collected from rabbits at different time points and used to determine antibody responses by ELISA (See results in Figures 19-21 ).
  • New Zealand White rabbits of 8 to 9 weeks of age were evaluated for antibody immune response using ELISA. Rabbits were fed food and water ad libitum and housed according to IUCUC standards and practices at Stillmeadow, Inc. (Sugarland, TX). Prior to the DNA vaccine administration, the injection site was shaved and thoroughly cleaned to remove excess hair and debris. On the day of DNA vaccination, rabbits were weighed, anaesthetized with ketamine/xylazine, and bled, and maintained on isoflourane (2%) for the duration of the treatment.
  • the plasmids encoding various Vaccinia virus antigens were administered (Day 0, 21, 42, and 84) either intradermally (ID) or intramuscularly (IM), in volumes of 100 ⁇ l and 500 ⁇ l, respectively, and consisted 1 mg of total plasmid per rabbit (125 ⁇ g per antigen and/or empty vector up to 1 mg per injection).
  • Figures 22 and 23 show the vaccination schedule and the vaccination parameters in detail.
  • Groups A to J The following combinations were used and were followed by electroporation (Groups A to J): Group A and F rabbits were immunized with an individual plasmid expressing a single antigen (B5R) antigen; Group B and G rabbits were immunized with a combination of four different antigens (A27L, B5R, H3L, and L1R); Groups C and H consisted of a combination of 8 plasmids expressing various antigens (A4L, A27L, A33R, A56R, B5R, F9L, H3L, and L1R); Groups D and I served as a negative control for antibody response and were immunized with the empty vector, pVAX1 (Invitrogen, Carlsbad, CA); and Groups E and J were vaccinated with the same combination of 8 antigens, but without electroporation. Each antigen formulation was prepared in 1% LGS.
  • All plasmids were administered into semimembranosus muscle followed by electroporation using the CELLECTRA ® constant current device (other than Group 5), at 0.5 Amps, 3 pulses, 52 ms/pulse, 1 sec between pulses for IM injections and 0.2 Amps, 4 pulses, 52 ms/pulse, 1 sec between pulses for ID injections.
  • Sera were collected from rabbits at different time points and used to determine antibody responses by protein ELISA ( Figures 24 to 33 ).
  • ELISA antigen preparation The antigens for ELISA were prepared by Abgent, Inc. (San Diego, CA). The ORF encoding the gene was PCR amplified using gene-specific primers containing appropriate restriction sites for cloning. The 3'-end oligonucleotide was designed to allow fusion with the 6X Histidine tag present in the prokaryotic expression vector, pEt21a(+). Proteins were purified using standard nickel column purification methods.
  • ELISA assays were performed by coating a MaxiSorp Immuno 96 well plate (Nunc, Rochester, NY) with 50ng of antigen (A4L, A27L, A33R, B5R, H3L, or L1R) diluted in PBS and incubated overnight at 4°C. Following washing with PBS supplemented with 0.05% Tween 20 (PBS-T), plates were blocked with PBS supplemented with 3% BSA and incubated for 1h at room temperature. Rabbit sera was diluted in PBS supplemented with 0.5% BSA; 0.05% Tween 20, and incubated (50 ⁇ l) overnight at 4°C.
  • antigen A4L, A27L, A33R, B5R, H3L, or L1R
  • the wells were incubated with the secondary antibody, goat anti-rabbit IgG-HRP conjugated (Sigma-Aldrich, St. Louis, MO) and diluted 1 in 10,000 in PBS supplemented with 0.5% BSA; 0.05% Tween 20 (100 ⁇ l/.well). The wells were incubated for 1h at room temperature and washed accordingly. TMB substrate and stop solution was added to each well according to manufacturer's recommendations (KPL, Gaithersburg, MD). Absorbance was measured at 450nm using the Lumistar Galaxy plate reader (BMG Labtech, Durham, NC).
  • ELISpot assay MultiScreen TM -IP 96 well plates (Millipore, Bedford, MA) were coated with monoclonal antibody (mAb) to monkey IFN- ⁇ (GZ-4) diluted 1 to 100 in PBS and incubated overnight at 4°C. After five washes with PBS, plates were blocked for 2 h at room temperature with complete culture medium (RPMI 1640 with 10%FBS, 1% Pennicilin/Streptomycin). PBMCs were added in triplicates at an input cell number of 2 X 10 5 cells in 100 ⁇ l of complete culture medium. Peptides were diluted in complete culture medium at a final concentration of 25 ⁇ g/ml and 100 ⁇ l dilution was added per well.
  • Concanavalin A (ConA, 5 ⁇ g/ml; Sigma-Aldrich, St. Louis, MO) was used as a positive control, and cells resuspended in complete culture medium only served as a negative control. After an incubation of 24 h at 37°C, plates were washed five times with PBS followed by overnight incubation at 4°C along with 100 ⁇ l/well of biotinylated detector mAb to monkey INF- ⁇ (7-B6-1) and diluted to 1,000 in PBS. Plates were washed and 100 ⁇ l/well of strepatavidin-alkaline phosphatase conjugate, diluted 1 to 1,000 in PBS were added and incubated at room temperature for 1 h.
  • the antigen specific IFN- ⁇ responses were calculated after subtraction of spots formed in control medium wells from the number of spots formed in response to the corresponding peptides used for stimulation. ELISpot assays were also performed after depletion of CD8 + lymphocytes from PBMCs by using CD8 depletion beads (Miltenyi Biotec, Gladbach, Germany).
  • DNA multivalent vaccine elicits a robust antibody response
  • Cynomolgus macaques were purchased from Three Springs Scientific (Perkasie, PA) and housed and cared for by Southern Research Institute (Birmingham, AL). A total of 24 macaques (14 females and 10 males) were individually housed in cages and assigned to each group based on similarity of body weights and sex. All macaques were tested negative for SIV, STLV, SRV, and HBV. Upon receipt, all animals were quarantined and acclimated to study rooms. Macaques were fed Teklad 2050C Diet during the quarantine and study periods. Feed was provided at a quantity of approximately one scoop of feed (6 to 10 biscuits) twice a day.
  • a multivalent DNA vaccine comprised of eight VACV Western Reserve strain genes: A4L, A27L, A33R, A56R, B5R, F9L, H3L, and L1R.
  • two groups of macaques were immunized with either a high dose or low dose vaccine by the intramuscular (IM) route.
  • One month following the third immunization the animals were challenged with a lethal dose of the Zaire 79 strain of monkeypox virus (introducing 2 x 10 7 PFU via intravenous delivery).
  • ELISA assays were used to evaluate the antibody-specific responses for each antigen in the multivalent DNA vaccine preparation (Table 3a ). Graphic representation of Table 3 can be seen in the bar graph depicted in Fig. 34a . Table 3a Antibody response measured for each antigens by ELISA.
  • Antibody responses are shown two weeks following the last vaccination. All antigens in the multivalent vaccine elicited an antibody response to varying degrees ( Table 3a ) irrespective of dosage and route of vaccination. A dose effect was observed for the antigens with the ID high dose (HD, 250 ⁇ g/plasmid), performing better in most cases than the low dose (LD, 125 ⁇ g/plasmid.) vaccine. For IM vaccination, antigen-specific responses were observed at both doses without a dose effect. In terms of responses, the ID route of delivery fared better than the IM delivery for the HD vaccine, with the exception of B5R which was equivalent.
  • Antigen-specific antibody titers were measured. Most antigens in the multivalent vaccine elicited an antibody response to varying degrees irrespective of dosage and route of vaccination. See results in Table 3b, below.
  • Table 3b Antigen-specific antibody titers Group Antigen pVAX1 IM-LD ID-LD IM-HD ID-HD A4 ⁇ 100 2751 750 ⁇ 100 2667 A27 ⁇ 100 7500 2667 7500 6709 A33 ⁇ 100 10000 10000 10000 7500 A56 ⁇ 100 7500 6250 9063 11667 B5 ⁇ 100 276 5017 1251 3334 F9 ⁇ 100 626 3001 2501 7500 H3 ⁇ 100 2501 3501 ⁇ 100 4167 L1 ⁇ 100 ⁇ 100 ⁇ 100 ⁇ 100 1668
  • the cellular immune response induced by the multivalent vaccine was also evaluated ( Fig . 34b ). Small differences in the level of responses were noticeable between the LD and HD for ID-EP, whereas a much more pronounced dosage effect was observed following IM-EP vaccination. A significant increase in the total cellular immunity followed both ID and IM injections, with robust immune response observed following the second and third vaccinations HD injections ( Fig. 34b ). An augmented effect was observed for ID-HD delivery with an overall increase of 8-fold (687 ⁇ 31.5 vs. 5675 ⁇ 538.1 per 10 6 SFU, P ⁇ 0.03) in cellular response. A similar effect was observed for IM-HD delivery. Between the first and second or third IM-HD vaccination we observed a 3-fold increase in cellular responses (2388 ⁇ 199 vs. 8028 ⁇ 719 or 7098 ⁇ 587 per 10 6 SFU, P ⁇ 0.02, respectively).
  • T cell functions were assessed by intracellular cytokine staining including: IFN ⁇ , IL-2, and TNF ⁇ production as well as CD107a, as a surrogate marker for degranulation.
  • IFN ⁇ IFN ⁇
  • IL-2 IL-2
  • TNF ⁇ TNF ⁇ production
  • CD107a CD107a
  • the functional T cell response to two antigens were analyzed: A27 and B5.
  • the overall magnitude of functional responses to A27 and B5 were higher in the CD4 + T cell compartment than the CD8 + T cell compartment.
  • the ID-HD group had the highest CD4 + T cell response with an average magnitude of 0.3 ⁇ 0.06% with all animals responding to at least one of the two antigens ( Figure 39a ).
  • the IM immunized animals had a lower average response although there was no significant difference in responses between the high and low dose group (0.2 ⁇ 0.08% and 0.2 ⁇ 0.1%, respectively).
  • the ID-LD group had the lowest CD4 + response (0.13 ⁇ 0.03%). In contrast to the CD4 + response, the CD8 + responses to A27 and B5 were slightly lower in magnitude ( Figure 39b ).
  • the IM-LD group had a response that was slightly higher than the ID-HD group (0.18 ⁇ 0.08 % and 0.15 ⁇ 0.04%, respectively). Both the ID-LD and IM-HD had modest CD8 + T cell responses (0.07 ⁇ .02% and 0.08 ⁇ .05%, respectively).
  • Another parameter of the cellular immune response is the proliferative capacity of the vaccine induced T cell response.
  • PBMCs were isolated following the third immunization and stimulated ex vivo, and then assessed for proliferation by CFSE dilution.
  • CD4 + T cell proliferation was highest in the ID-HD group (10.2 ⁇ 6.2%) ( Figure 40a ).
  • the ID-LD and IM-HD groups had a low CD4 + T cell response of (1.7 ⁇ 0.67% and 1.4 ⁇ 1.1%, respectively).
  • the IM-LD did not have a response that was higher than background. Similar results were seen in the CD8 + T cell compartment with the ID-HD group having the highest response (6.7 ⁇ 5.4%) ( Figure 40b ).
  • the ID-LD, IM-HD, and IM-LD groups had similar levels of CD8 + T cell proliferation (1.6 ⁇ 0.69%, 1.9 ⁇ 1.1%, and 1.8 ⁇ 1.6%, respectively).
  • CHALLENGE vaccinated nonhuman primates and challenge with monkeypox Zaire 79
  • Multivalent DNA vaccine protects nonhuman primates from severe monkeypox disease
  • the vaccine substantially reduced the number of lesions and provided 100% protection from mortality at both low and high doses, compared to the innumerable number of lesions and 75% mortality recorded in the pVAX1-treated control group.
  • 3 of the 4 animals in the pVAX1-treated animals had innumerable lesions; the disease was fatal for the 4 th animal.
  • the lesions started to resolve by day 15, with the effect more pronounced for the animals receiving ID-HD injection: at day 21, only one of the six animals had lesions (with only 4 lesions observed).
  • the detection of antibodies induced upon DNA vaccine and monkeypox virus challenge was measured by ELISA using purified, inactivated whole VACV as a coating antigen ( Fig. 36 ).
  • Low level titers of virus-specific VACV-specific antibodies were detected on day 28 in animals that received the DNA vaccine, with all animals having a 1:100 endpoint titer for both vaccination groups and dosages.
  • Antibody titers started to increase thereafter with average endpoint titers of 1:633 and 1:300 on day 91 for ID-HD and IM-HD delivery routes, respectively, with insignificant differences existing between dosages. Dramatic increases in anti-VACV antibody titers were observed post-challenge in vaccinated macaques.
  • the pVAX1-treated animals experienced significant weight loss, elevated body temperature, depression, and lethargy during the acute phase of the infection. The most significant weight loss was observed on day 12 post-challenge when pox lesions and viremia peaked (Table 6). Table 6. Bodv weight changes following intravenous challenge with monkeypox virus. Day post-challenge* Vaccine Macaque No.
  • animals vaccinated via the IM route experienced a weight gain of 1.7% over pre-challenge weight.
  • the animals vaccinated with the LD vaccine experienced a greater weight loss that that observed for the HD-vaccinated animals.
  • Vaccinated animals also had elevated WBC numbers to the same extent on day 12 with an average percent increase of 69.4 ⁇ 15.8, 68.8 ⁇ 15.6, 80.9 ⁇ 17.2, 70.0 ⁇ 28.0 for ID-LD, ID-HD, IM-LD, IM-HD, respectively, over pre-challenge levels.
  • most vaccinated animals from each group had their WBC numbers return to pre-challenge levels, whereas the surviving macaque from the control group continued to have elevated WBC number of 22,300 per ⁇ L.

Claims (16)

  1. Vaccin à ADN susceptible de produire une réponse immunitaire protectrice chez des mammifères contre un poxvirus comprenant :
    un plasmide d'ADN comprenant une séquence de nucléotides qui code l'antigène VACV A27L,
    un plasmide d'ADN comprenant une séquence de nucléotides qui code l'antigène VACV F9L,
    un plasmide d'ADN comprenant une séquence de nucléotides qui code l'antigène VACV H3L,
    un plasmide d'ADN comprenant une séquence de nucléotides qui code l'antigène VACV L1R,
    un plasmide d'ADN comprenant une séquence de nucléotides qui code l'antigène VACV A33R,
    un plasmide d'ADN comprenant une séquence de nucléotides qui code l'antigène VACV A56R,
    un plasmide d'ADN comprenant une séquence de nucléotides qui code l'antigène VACV B5R, et
    un plasmide d'ADN comprenant une séquence de nucléotides qui code l'antigène VACV A4L.
  2. Vaccin à ADN susceptible de produire une réponse immunitaire protectrice chez des mammifères contre un poxvirus, comprenant :
    un plasmide d'ADN codant chacun des antigènes VACV MV A27L, F9L, H3L et L1R ;
    un plasmide d'ADN codage chacun des antigènes VACV EV A33R, A56R et B5R ; et
    un plasmide d'ADN codant l'antigène A4L.
  3. Vaccin à ADN selon la revendication 1, comprenant huit plasmides d'ADN distincts, dans lequel un plasmide d'ADN comprend une séquence de nucléotides qui code l'antigène VACV A27L, un plasmide d'ADN comprend une séquence de nucléotides qui code l'antigène F9L, un plasmide d'ADN comprend une séquence de nucléotides qui code l'antigène VACV H3L, un plasmide d'ADN comprend une séquence de nucléotides qui code l'antigène VACV L1R, un plasmide d'ADN comprend une séquence de nucléotides qui code l'antigène VACV A33R, un plasmide d'ADN comprend une séquence de nucléotides qui code l'antigène VACV A56R, un plasmide d'ADN comprend une séquence de nucléotides qui code l'antigène VACV B5R et un plasmide d'ADN comprend une séquence de nucléotides qui code l'antigène VACV A4L.
  4. Vaccin à ADN selon n'importe laquelle des revendications 1 à 3, dans lequel lesdits plasmides d'ADN comprennent des séquences consensus d'ADN qui codent lesdits antigènes.
  5. Vaccin à ADN selon n'importe laquelle des revendications 1 à 4, dans lequel :
    l'antigène A4L a la séquence d'acides aminés SEQ ID n° 2, l'antigène A27L a la séquence d'acides aminés SEQ ID n° 4, l'antigène B5R a la séquence d'acides aminés SEQ ID n° 6, l'antigène A33R a la séquence d'acides aminés SEQ ID n° 8, l'antigène A56R a la séquence d'acides aminés SEQ ID n° 10, l'antigène F9L a la séquence d'acides aminés SEQ ID n° 12, l'antigène H3L a la séquence d'acides aminés SEQ ID n° 14, et l'antigène L1R a la séquence d'acides aminés SEQ ID n° 16.
  6. Vaccin à ADN selon n'importe laquelle des revendications 1 à 5, dans lequel :
    l'antigène A4L est codé par SEQ ID n° 1, l'antigène A27L est codé par SEQ ID n° 3, l'antigène B5R est codé par SEQ ID n° 5, l'antigène A33R est codé par SEQ ID n° 7, l'antigène A56R est codé par SEQ ID n° 9, l'antigène F9L est codé par SEQ ID n° 11, l'antigène H3L est codé par SEQ ID n° 13, et l'antigène L1R est codé par SEQ ID n° 15.
  7. Vaccin à ADN selon n'importe laquelle des revendications 1 à 6, dans lequel ledit vaccin à ADN comprend des plasmides d'ADN pGX4001 (figure 2), pGX4002 (figure 5), pGX4003 (figure 4), pGX4004 (figure 5), pGX4005 (figure 6), pGX4006 (figure 7), pGX4007 (figure 8) et pGX4008 (figure 9).
  8. Vaccin à ADN selon n'importe laquelle des revendications 1 à 7, dans lequel chacun desdits plasmides d'ADN est présent à une dose plus grande que 50 µg.
  9. Vaccin à ADN selon n'importe laquelle des revendications 1 à 7, dans lequel chacun desdits plasmides d'ADN est présent à une dose de 125 µg.
  10. Vaccin à ADN selon n'importe laquelle des revendications 1 à 9, comprenant en outre un adjuvant sélectionné parmi IL-8, IL-12, IL-15, IL-18, IL-28, MCP-1, MIP-Iα, MIP-1p, RANTES, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, CTACK, TECK, ou MEC, ou une combinaison de ceux-ci.
  11. Vaccin à ADN selon n'importe laquelle des revendications 1 à 10, dans lequel ledit vaccin à ADN est susceptible de produire une réponse immunitaire protectrice chez des mammifères contre un virus de la variole.
  12. Vaccin à ADN selon n'importe laquelle des revendications 1 à 10, à utiliser pour induire une réponse immunitaire protectrice chez un mammifère à un poxvirus, incluant une réponse d'anticorps de neutralisation.
  13. Vaccin à ADN à utiliser comme revendiqué dans la revendication 12, dans lequel le vaccin à ADN est pour une injection intradermique ou intramusculaire.
  14. Vaccin à ADN à utiliser comme revendiqué dans n'importe laquelle des revendications 12 et 13, dans lequel l'incitation le fait d'induire une réponse immunitaire protectrice comprend l'injection du vaccin à ADN dans un tissu dudit mammifère, et comprenant en outre l'étape d'électroporation dudit tissu avec une certaine quantité d'électroporation d'énergie électrique.
  15. Vaccin à ADN à utiliser comme revendiqué dans la revendication 14, dans lequel l'étape d'électroporation comprend la délivrance d'un courant constant audit tissu, de manière facultative la délivrance d'un courant de 0,2 A.
  16. Vaccin à ADN à utiliser comme revendiqué dans n'importe laquelle des revendications 14 et 15, dans lequel le fait d'induire une réponse immunitaire protectrice comprend en outre la répétition de ladite étape d'injection.
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